LOCUS       sequence-305926-        3857 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cell-free expression vector for Zika virus NS2B protein with C-term 
            mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3857)
  AUTHORS   Varasteh Moradi S, Gagoski D, Mureev S, Walden P, McMahon KA, Parton
            RG, Johnston WA, Alexandrov K
  TITLE     Mapping Interactions among Cell-Free Expressed Zika Virus Proteins.
  JOURNAL   J Proteome Res. 2020 Apr 3;19(4):1522-1532. doi: 
            10.1021/acs.jproteome.9b00771. Epub 2020 Mar 9.
  PUBMED    32081002
REFERENCE   2  (bases 1 to 3857)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1021/acs.jproteome.9b00771"; journalName: "J Proteome Res"; 
            date: "2020-04-3- 3"; volume: "19"; issue: "4"; pages: "1522-1532"
COMMENT     Sequence Label: pCellFree_G06_ZIKV NS2B
FEATURES             Location/Qualifiers
     source          1..3857
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..26
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7..25
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    160..184
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(578..602)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             604..627
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(764..782)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1032..1051
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1336..1359
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     terminator      1582..1628
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(1706..1722)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1706..1722)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1719..1741)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1730..1746
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(1754..1760,1761..1778,1779..1784))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1799..1820
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1937..1954)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2108..2696)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2188..2207)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2867..3658,3659..3727))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERTRQIAEIGAS
                     LIKHW"
     primer_bind     3490..3509
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3728..3832)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtctaat acgactcact atagggacat cttaagttta ttttatttta ttttatttta
       61 ttttatttta ttttatttta ttttatttta ttttatttaa ccatgacagt aatgtataaa
      121 gtctgtaaag acattaaaca cgtaagtgaa accatgggca caagtttgta caaaaaagca
      181 ggcttcagct ggccccctag tgaagttctc acagccgttg gcctgatatg tgcactggcc
      241 ggagggtttg ccaaggcaga cattgagatg gctggaccga tggctgcagt aggcttgcta
      301 attgtcagct atgtggtctc gggaaagagt gtggacatgt acattgaaag agcaggagac
      361 atcacatggg aaaaggacgc ggaagtcact ggaaacagtc ctcggcttga cgtggcactg
      421 gatgagagtg gtgacttctc cttggtagag gaagatggtc cacccatgag agagatcata
      481 ctcaaggtgg tcctgatggc catctgtggc atgaacccaa tagctatacc ttttgctgca
      541 ggagcgtggt atgtgtatgt gaagactggg aaaaggaacc cagctttctt gtacaaagtg
      601 gtgctggagg tgctcttcca gggcccgaag ggcgaggagg ataacatggc catcatcaag
      661 gagttcatgc gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc
      721 gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc
      781 aagggtggcc ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc
      841 aaggcctacg tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag
      901 ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag
      961 gactcctccc tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc
     1021 ccctccgacg gccccgtaat gcagaagaag actatgggct gggaggcctc ctccgagcgg
     1081 atgtaccccg aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac
     1141 ggcggccact acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg
     1201 cccggcgcct acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc
     1261 atcgtggaac agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg
     1321 tacaagacct ctggtcacca ccatcaccac catcaccact aagcggccgc cctcctcctc
     1381 ctttcttgtt cctttcacgt cgccttctcg gttgtagctg gcagacgacg agtcttactt
     1441 ttacgtgtac ttctctatag atgatgtatg atctctctgc atgcgtgttc gtgcatgtgt
     1501 ccgtgtgttg ggtacgcgtg gtaccctgca ggaaggaagc tgagttggct gctgccaccg
     1561 ctgagcaata actagtaatt actagcataa ccccttgggg cctctaaacg ggtcttgagg
     1621 gggttttttg ctgaaaggag gacagctgat gattgtcatg cttgccatct gttttcttgc
     1681 aaggtcagag gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     1741 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     1801 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     1861 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     1921 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     1981 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     2041 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     2101 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     2161 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     2221 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     2281 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     2341 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     2401 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     2461 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     2521 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     2581 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     2641 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagagg
     2701 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     2761 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     2821 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     2881 tcagtgaggc acctatctca gcgatctgtc tagttcgttc atccatagtt gcctgactcc
     2941 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3001 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3061 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3121 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3181 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3241 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3301 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3361 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     3421 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     3481 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     3541 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     3601 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     3661 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     3721 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     3781 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     3841 cccgaaaagt gccacct
//
